Methods and apparatus for the quantitation of nuclear proteins

ABSTRACT

An image analysis system is used for the quantitation of nuclear proteins in cell populations. Particularly, the hormonal receptor content of fine needle aspirates of human breast carcinomas are evaluated. Estrogen or progesterone receptors are amplified and visualized in the specimen by a staining technique of the immunoperoxidase type. Monoclonal antibodies specific against the receptor are attached to the receptor sites and are then amplified by a bridging antibody which attaches to the monoclonal antibody and a peroxidase-antiperoxidase complex. A chromagen, diaminobenzidine is combined with the complex and treated with hydrogen peroxide to react with the peroxidase forming an insoluble brown precipitate which marks the receptor sites for optical identification. The specimen is then counterstained with another chromagen, methyl green which is specific to the nucleus of each cell. Two monochromatic filterings optionally separate the areas stained by the receptor site optical enhancer and the nuclear area optical enhancer. Measurements of the optical density values of the stained receptor areas yield an intensity value directly related to the quantity of hormonal receptor in the specimen. A comparison of the nuclear area containing hormonal receptor with the total nuclear area yields a percentage value which indicates the distribution of cells throughout of the specimen which contain receptor. These two values for intensity and distribution are then combined to yield a predictive score for an assay. The measured score when compared to an empirically derived reference score is predictive of the prognosis of endocrine therapy.

This application is a continuation-in-part of patent application Ser.No. 794,937, filed Nov. 4, 1985 and, now U.S. Pat. No. 4,741,043.

The invention pertains generally to methods and apparatus for thequantitation of nuclear proteins, and is more particularly directed tothe quantitative assay of nuclear material by image analysis which canbe visualized with image enhancement factors specific to the proteinunder assay, such as monoclonal antibodies against the estrogen andprogesterone receptors of carcinomas, specifically identifiable types ofhuman breast cancers, or the like, bound to a peroxidase-antiperoxidasecomplex.

Nuclear proteins are of considerable interest in the medical fieldtoday. Their identification and the mapping of their structure are notonly providing a view into the inner workings of cells, and possiblylife itself, but are also useful in exploring the development and cureof particular pathologies. One of the more promising areas which willgreatly benefit mankind is the research being done in nuclear proteinanalysis in relation to human carcinomas.

Specifically, it has been estimated that one out of every eleven womenin the United States will develop some type of breast cancer in herlifetime. In spite of major advances during the last decade inchemotherapy and hormonal therapy for these diseases, the death ratefrom such disease has remained substantially the same. It is stronglysuggested that further characterization and quantitation of the cancercells responsible for these diseases is necessary to provide better andnew methods of treatment.

It has long been known that some human breast cancers arehormone-dependent, such that they undergo striking regression whendeprived of a supporting hormone by removal of the ovaries, adrenals, orpituitary; or an altering the hormonal milieu by the administration ofandrogens, large doses of estrogen, or antiestrogens such as tamoxifen.For those patients who respond, endocrine manipulation by eitherablative or additive means may represent the best treatment nowavailable for advanced breast cancer. However, only 25-30% of cancerpatients have breast tumors of the hormone-dependent type. There hasbeen a need for a means to distinguish those individuals who arefavorable candidates for endocrine therapy, from the larger group whosebreast cancers are unresponsive to hormonal treatment and who should beplaced directly in chemotherapy.

Researchers have found that breast cancers showing low estrogen bindinglevels or lacking cytosolic estrogen receptor rarely respond toendocrine therapy, whereas most but not all patients with receptorcontaining tumors receive objective benefit from endocrine treatment. Asthe sensitivity of methods for the detection and estimation ofestrophilin has improved, it has become apparent that some cancersthought to lack receptors actually contain very low levels of thisprotein. However, these tumors do not respond to endocrine therapy andshould be classified with those totally lacking receptor. Therefore, thequalitative classification of "receptor-positive" or "receptor-negative"for tumors must be replaced with quantitative terms such as"receptor-rich" or "receptor poor" to predict favorable results, withthe dividing line determined empirically. More recently, researchershave discovered that progesterone levels in tumors provide similarindications to those of estrogen levels. There remains the problem ofaccurately and reproducibly quantitating estrogen and progesteronereceptor levels from tissue samples to determine where a tumor should beclassified.

To be able to accurately quantify the estrogen receptor content in atissue it has in the past been necessary to perform a biochemical assayon the tissue sample. One of the most used methods is the sucrosedensity gradient configuration as described by Jensen, et al. "HormoneDependency in Breast Cancer" J. Steroid Biochem. 7:911-917, 1976.Biochemical assays have the problem of being extremely complex, timeconsuming, and expensive to accomplish along with the problem that theyare not generally done at the same institution which makes a biopsy ofthe tumor.

Such biochemical assays also must use relatively large amounts of tissueand they consume the tissue sample. Additionally, biochemical assays candiffer inexplicably between samples of the same tumor. While it isbelieved this may be due to the heterogeneity of the tissue samples,even in a single tumor there is no sure way to separate differentfeatures because a biochemical assay averages the estrogen receptorcontent over all the cellular material assayed. A sample with a mix oftumor cells and regular cells will be represented quite differently thana sample with all tumor cells, even though both samples really containthe same estrogen receptor content.

To partially overcome some of these difficulties, others in the art havesought to provide histochemical methods for the visualization of thecontent of estrogen receptors in a specimen by tagging the receptorswith markers which can then be measured independently. Researchers haveused radiolabeled estrogen, fluorescent estrogens, and conjugatedanti-estradiol antibodies which are only relatively specific to thereceptors. These tags can then be visualized by radiating the tissuesample with the correct beam energy such as X-rays or a particularfrequency of light. Generally, these attempts at histochemicallocalization of estrogen receptor with anti-steroid antibodies orfluoresceinated estrogens have not yielded acceptable sensitivities andspecificities when compared to the previous biochemical assays.

A newer immunohistochemical technique with much greater promise has beendeveloped as a staining technique using a peroxidase-antiperoxidasecomplex, see King et al., "Comparison of Immunocytochemical andSteroid-Binding Assays for Estrogen Receptor in Human Breast Tumors"Cancer Research, V45, pgs. 293-304, January 1985. The method comprisesutilizing a highly specific monoclonal antibody directed against theestrogen receptor protein. Such monoclonal antibodies developedspecifically against estrogen receptor include those first characterizedat the University of Chicago and designated H222 sP2 and H226 sP2. Theprincipal advantages of this approach over the fluorescein-taggedligands and radioisotope localization are the availability ofwell-characterized reagents and a sensitive immunoperoxidase techniquefor amplification and detection of the small amounts of receptormolecules present in a tissue sample.

The advantages of the PAP staining technique over a biochemical assayare greater, in that only a small tissue sample need be used and suchsamples can be taken from a patient through a fine needle aspiration.Thus, the immunocytochemical assay can be accomplished on tumors toosmall to yield sufficient material for biochemical assays. Because theindividual cells of the specimen are visualized, heterogeneity in tissuesamples can be dealt with adequately. In general, the immunocytochemicalassays are simpler, less expensive, and more accurate than conventionalbiochemical assays.

Another reason for the difficulty in producing quantitative resultswhich can be duplicated relates to the substantial lability of theestrogen receptor to partial or complete loss of its ability to bindestrogen under very mild conditions. There is reason to believe that theantigenic site of the receptor may be more stable to degradation than itis to binding activity. Thus, the immunohistochemical technique whichcan be accomplished shortly after an excision or biopsy has been made ispreferable to the usual delay before a biochemical assay can be run.

For a researcher to determine the estrogen receptor content with the newimmunohistochemical method, he views a stained tissue sample through amicroscope and makes a subjective visual determination of the amount ofstaining for each cell. A weighted average of the observed cell stainingis then used to form an opinion whether the specimen is receptor rich orreceptor poor. This method provides but a qualitative, or at best asemi-quantitative, immunohistochemical technique for the assessment ofestrogen receptor status in human breast cancers.

The data obtained by this method does indicate a significant correlationbetween immunohistochemical assays and quantitative biochemical assays,see McCarty, Jr. et al., "Estrogen Receptor Analysis", Arch Pathol LabMed, Vol 109, August 1985. One researcher, Pertschuk, reported theresults of 43 patients and McCarty Jr. et al. reported the results of 23patients with metastatic breast carcinoma who were treated with hormonaltherapy. The positive predictive value of the immunocytochemical assayin these patients was 85% and the negative predictive value was 91%. Thecorresponding values for conventional steroid-binding assays in thesecases was 69% and 79%, respectively. In this limited number of patients,the immunocytochemical assay had a stronger correlation to the responseto endocrine manipulative management than did the conventionalsteroid-binding assays.

However, the remaining problem of immunohistochemical assays relative toconventional methods is the lack of an accurate and reproduciblequantitative assay at the cellular level. It is believed the developmentof a true quantitative assay at the cellular level using theimmunohistochemical technique may further raise the positive predictivenature of the assay as a prognosis of endocrine therapy.

SUMMARY OF THE INVENTION

The invention provides methods and apparatus for the measurement of thequantity of specific nuclear proteins in a cellular population. Onepreferred method includes the utilization of computerized image analysisto determine from a digital grey scale image of a cell population thequantity of a particular protein. The grey scale image is representativeof the amount of an optical enhancement factor, such as a chromogen,which binds to an equivalent amount of the specific protein under studyand thereby allows optical amplification and visualization of theprotein.

The apparatus includes a means for magnifying and displaying an image ofa group of cells of a specimen from a field on a microscope slide. Thespecimen cell population is prepared with a special staining andcounterstaining technique. After staining, the image field is digitizedby the apparatus and stored in a memory provided by the apparatus. Fromthe digitized image, a nuclear image mask is formed by filtering theimage at one wavelength of light. The nuclear mask is stored and asecond filter is used to form another filtered image of the areas withthe optical enhancement factor. Differentiation of cellularcharacteristics can be made by comparing the first image with the secondto obtain a quantitation of material stained with the opticalenhancement factor and thus, an assay of the amount of the particularnuclear protein which is under study.

In a preferred embodiment, the invention is used to assay the tumorspecimens of human breast cancers for hormonal receptor content,particularly estrogen and progesterone receptors. The estrogen andprogesterone receptor content of breast carcinomas have been shown toprovide clinically useful data in the medical management of patientswith breast cancers. Endocrine manipulative management techniques canuse the measured levels as predictors of the favorable use of thesetherapies.

Moreover, the methods of the invention for providing quantitativeimmunohistochemical assays for estrogen and progesterone receptorcontent can be applied to quantitative assays of other nuclear proteins,especially oncongene protein products (c-myb, c-myc, n-myc, c-fos), atleast one of which (n-myc) has already been clinically shown to beimportant in determining the prognosis of neuroblastoma andretinoblastoma.

For estrogen receptor assays, specimens are prepared using acounterstaining technique where in a first step, the specimen is stainedusing an immunoperoxidase staining technique, the peroxidase techniqueincluding the use of a monoclonal antibody to estrophilin. In a secondstep, the specimen is counterstained with another optical enhancementfactor, preferably methyl green. The resulting preparation has greennuclei with varying degrees of brown diaminobenzidine (DAB) precipitatelocalized in the nuclei with the estrogen receptor. While acounterstaining technique using peroxidase and methyl green isexemplary, other stains and optical enhancement factors are availablewhich can be conjugated or attached to a particular antigen and theinvention is not limited to only the example shown.

However, spectral studies have shown that the methyl green stain offersgood spectral separation from the diaminobenzidine precipitate of theimmunoperoxidase technique so that different features of the image canbe readily separated by filtering it at two different wavelengths. Thisallows the image to be digitized into two separate images, one in whichall the cell nuclei are optically enhanced (methyl green), and one inwhich only those nuclear areas with receptor staining (DAB) areoptically enhanced. In a preferred embodiment, the images can beseparated by a 650 nanometer (red) filter to produce an image of all ofthe nuclei, and a 500 nanometer (green) filter to produce an image ofonly those nuclear areas with the diaminobenzidine precipitate staining.

To further differentiate those areas an interactive threshold settingtechnique is provided where an operator visualizing the images can set aboundary on the areas under consideration. When the boundaries are set,the images are formed by eliminating all parts of the image which arebelow the thresholds in optical density. A threshold, termed a nuclearboundary, is set for the first image, and a threshold, termed anantibody boundary, is set for the second image.

The image processing method then consists of first forming the maskimage of the nuclei under consideration with the red filter. This maskimage is stored and another image for the estrogen quantitation is thenacquired by using the green filtered version of the same image. Theeffect of the filters in combination is to optically enhance (makedarker) those areas of the nuclear mask where nuclei are stained withdiaminobenzidine and to make lighter those nuclei with only methyl greencounterstain. An image analysis can then be performed using only thoseareas of the image which are stained and which are within the mask.

Another aspect of the invention provides a means for determining whicharea of an image field is to be measured. A window or box is displayedon the first image where the window can be moved and varied in size.This allows for the selection of particular cells to be included in thepopulation measured and for other cells to be excluded. In this manner,the operator of the apparatus can distinguish normal cells fromcarcinoma cells so that a heterogeneous specimen can be measured moreaccurately. The feature also allows for the exclusion of debris,necrotic tissue, blood cells, etc. from the image analysis.

Statistical data of the differences between, and comparison of the twoimages such as a histogram may be used to quantitate the amount ofestrogen receptor in the cell population under study. Also, theproportion or percentage of total nuclear area stained may be easilymeasured as the area stained above an antibody threshold level in thesecond image.

With the ability to assay not only the intensity of the estrogenreceptors but also their distribution in a cell population, a method isprovided by the invention for predicting favorable endocrine therapyresponse based upon a combination of these factors.

A cell population is measured with the apparatus of the invention todetermine the percentage of positive stained cells in the population andtheir average stain intensity. A combination score of these two factorsis made according to the formula: ##EQU1## where QIC=a quantitativeimmunocytochemical score; and

N=a scaling factor.

It has been determined empirically that when the scaling factor is 10, aQIC score of ≧18 corresponds with excellent sensitivity and specificityto other quantitative and semi-quantitative estrogen receptor assays.Particularly, a QIC score of ≧18corresponds with a 98% sensitivityfactor and a 100% specificity factor when compared to a biochemicalassay of estrogen receptor setting an estrogen receptor rich thresholdat 10 Fmol./mg. of cytosol.

For progesterone receptor assays, the methods and apparatus are the sameas previously described with only specimen preparation being different.The specimen is stained using an immunoperoxidase staining techniqueincluding the use of a monoclonal antibody against progesterone.

Accordingly, it is an object of the invention to provide methods andapparatus useful in performing a quantitative image analysis forcharacterizing nuclear protein content, particularly steroid hormonereceptors of small cytological samples and especially fine needleaspirates of breast carcinomas.

It is another object of the invention to provide an image analysis of acell population capable of quantitating estrogen receptor orprogesterone receptor of human malignancies of the breast and femalegenital tract.

Still another object of the invention is to provide image analysismethods useful in predicting the responsiveness of cancer patients toendocrine manipulative management.

Yet another object of the invention is to provide a quantitativemeasurement value of hormonal receptor which is useful in predictingestrogen or progesterone rich carcinoma samples.

These and other objects, features, and aspects of the invention willbecome more apparent upon reading the following detailed descriptionwhen taken in conjunction with the attached drawings wherein:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pictorial representation of an image analysis systemconstructed in accordance with the invention;

FIG. 2 is a functional block diagram of the image analysis systemillustrated in FIG. 1 which is adapted to perform the methods for thequantitation of nuclear proteins in accordance with the invention;

FIG. 3 is a functional system diagram illustrating the major operationsof the system control illustrated in FIG. 2;

FIGS. 4 and 5 are perspective and cross-sectional views, respectively,of a slide particularly adapted for use in the image analysis systemillustrated in FIG. 1 having separate areas for calibration cell objectsand specimen cell objects;

FIG. 6 is an enlarged perspective view of a one embodiment of a filterselection apparatus for the image analysis system illustrated in FIG. 1;

FIG. 7 is a pictorial view at the microscopic level of the bindingeffects of a monoclonal antibody against estrogen receptor sites, abridging antibody, and a peroxidase-antiperoxidase complex;

FIG. 8 is a pictorial view at the microscopic level of the bindingeffects of a control monoclonal antibody (immunoglobulin) againstnonspecific receptor sites;

FIG. 9 is a graphical representation of the % of light transmission as afunction of light wavelength for the two counterstains and the two colorfilters used in accordance with the invention;

FIG. 10 is a functional flow chart of one preferred method ofquantitating hormonal receptor for human carcinoma in accordance withthe invention;

FIGS. 11, 12, and 13 are pictorial representations of images of a cellpopulation showing an unfiltered image, a red filtered image, and agreen filtered image, respectively;

FIG. 14. is a pictorial representation of the nuclear mask screen whichappears on the instruction monitor illustrated in FIG. 1;

FIG. 15 is a pictorial representation of the antibody quantitationscreen which appears on the instruction monitor illustrated in FIG. 1;

FIG. 16 is a system flowchart of the analysis screen architecture of theimage analysis system illustrated in FIG. 1;

FIG. 17 is a functional flow chart of the main menu of the initialscreen illustrated in FIG. 12;

FIG. 18 is a functional flow chart of the label/calibrate menu of thelabel/calibrate screen illustrated in FIG. 12;

FIG. 19 is a functional flow chart of the nuclear mask menu of thenuclear mark screen illustrated in FIG. 12;

FIG. 20 is a functional flow chart of the adjust nuclear boundary menuof the adjust nuclear boundary screen illustrated in FIG. 12;

FIG. 21 is a functional flow chart of the antibody quantitation menu ofthe antibody quantitation screen illustrated in FIG. 12; and

FIG. 22 is a functional flow chart of the adjust antibody boundary menuof the adjust antibody boundary screen illustrated in FIG. 12.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

With reference to FIGS. 1 and 2 of the drawings, the invention isembodied as an apparatus 11 (FIG. 1) which functionally operates as adigital image analysis and processing system 13 (FIG. 2). The apparatus11 comprises a high resolution microscope 15 with which an operator canview specimens on a support, in a preferred embodiment a glass slide 14.The microscope 15 has adjustment means 70 for focusing its optics 16 onthe slide 14 and a platform 51 movable incrementally in the X and Ydirections by positioning means 12 and 17 in order to view differentareas thereof. Positioning means 12, 17 and 70 in the form of adjustmentverniers are conventional for instrument quality microscopes.

The specimens in the field under study are further viewable by theimaging system 13 via a television camera 18. The camera 18 views thelight intensities of the image of the field and converts them into ananalog signal which can be sampled and processed by the imaging system13. The image analysis system 13 is controlled by a system control 22 inthe form of a digital processor such as a personal computer.

An operator can interactively communicate with the system control 22 viaa keyboard 36, and interacts further with the system to quantitatenuclear proteins by the viewing of two displays or monitors. A firstdisplay, image monitor 37, is a conventional RGB monitor which displaysthrough the system control 22 and camera 18, the same image field asseen through the microscope 15. A second display, instruction monitor62, is another conventional RGB monitor and is used to provide theoperator with interactive prompts, messages, information, andinstruction screens from a system program executed by the system control22.

The keyboard 36 is preferably an AT type keyboard which has on theleft-hand side a plurality of function keys F1-F10, in the middle aplurality of alphanumeric keys including the special keys of ENTER,SHIFT, CONTROL, and ALTERNATE, and on the right-hand side cursormovement keys including up, down, left and right arrow keys, a numerickeypad, a number lock key, and an escape key. A keyboard interface 35translates keystrokes into numerical codes recognized by the systemcontrol 22 for specific key indications. A printer 38 is provided forproducing reliable hard copy output of the data produced by theapparatus 11.

A functional schematic of the apparatus 11 is illustrated in FIG. 2 asimage analysis and processing system 13. The image processing system 13is used to analyze a plurality of specimen cell objects on the supportor glass slide 14 of the microscope 15. Suitable high resolutionmicroscope optics 16 receive light from a variable intensity source 19and transmit the light through the slide 14. The light is then passedthrough a filter selection apparatus 23 having three selectable filters.An optical image of each of the cell objects on the slide 14 passesthrough the filter apparatus 23 to an image splitter 25 which can takethe form of a prism.

On one side of the splitter 25, the television camera 18, or otherdetector, converts the optical images point by point into a scannedelectronic signal representing the optical intensity of each point inthe image. The output of the camera 18 which is formatted as a standardNTSC analog video signal is applied to an analog to digital converter ofan image processing interface 21. The image processing interface 21samples the analog signal and converts the image signal from thetelevision camera 18 to a digitized signal which is received and storedby the system control 22. Because of continuous scanning, a real timeimage of the area the optics 16 are focused on is provided by the imagedisplay 37. In general, the digital image is stored as a 512×512 arrayof pixels each having a measured light intensity of 0-255 (eight bits).

Because the source 19 transmits light through the cell objects on slide14, the optical density of each pixel of the image will convert thelight into a different intensity depending upon its percentage oftransmission. Areas with no cell objects in them will appear relativelylight or intense and areas having nontransmissive objects will appeardarker. In general, unmodified cell objects are relatively transparentand their features difficult to distinguish. Staining the cell objectsallows an optical enhancement of the features stained so they willappear darker than other features and their background. The inventionenhances a specific nuclear protein, either estrogen or progesterone, bystaining to permit its visualization by the image analysis system 13.

On the other side of the image splitter 25 are located the viewingoptics 24 of the microscope 15. This parfocal arrangement allows thesame image seen in the viewing optics 24 to be displayed on the imagedisplay 37. This feature allows the positioning of the platform 51 bythe manual X, Y adjustment of positioning means 12 and 17 until theoperator views a field of interest on the slide 14. At that time, acomputer enhanced digitized image of the selected field is displayed onthe image display 37 for further analysis. An X position sensor 26 and aY position sensor 27 generate position signals to a position interface34 which digitizes these signals to provide the apparatus 11 with anaccurate coordinate representation of the field in view.

Both of the displays 37 and 62 are controlled by the system control 22through standard video monitor interface circuitry 39 and 61,respectively. Similarly, the keyboard 36 and the printer 38 communicatewith the system control 22 through conventional interface circuitry 35and 41, respectively. In addition, the system control 22 controls arandom access memory 73 and bulk memory storage in the form of eitherfloppy and hard disk drives 75 through a memory control interface 71.

All of the interface circuits 21, 34, 35, 39, 41, 61, and 71 can beselectively embodied on printed circuit boards which are mounted in thebackplane or card connector of a conventional personal computer formingthe system control 22. Preferably, the personal computer can be onemanufactured by the IBM Corporation having a model designation AT. Suchsystem control 22 can be run under a disk operating system such as PCDOS, version 3.1 or later. The system software for the image analysis iscalled as an application program from the disk drive 75, and could, forexample, be supplied on a floppy disk 77. The system software is readfrom disk 77 and loaded into RAM 73. After loading, program control istransferred to the analysis software to regulate the various hardwareelements of apparatus 11 previously set forth in a known manner.

The image analysis system 13 operates under an interactive programcontrol by providing a number of instruction screens or images on theinstruction monitor 62 to assist the operator in the quantitation ofnuclear proteins, i.e. estrogen receptors or progesterone receptors,found in one or several cell populations displayed on image monitor 37.Through interactive responses by the operator and menu selections ondifferent instruction screens, the basic system functions of the imageanalysis are performed.

The system functions are more fully illustrated in FIG. 3 where softwarecontrol logic functions for the hardware in block 80 are showncommunicating with software analysis and measuring functions of thesystem program in blocks 82-96. Software is included in the systemprogram to perform an initialization and an interfacing of the operatingsystem functions and overall control of the instrument by instrumentcontrol logic. A screen handler for the instruction screens and thevideo display of the specimen digital images is performed for both ofthe monitors by image and instruction monitor control logic. The memoryand disk storage are handled in the software by memory control logic.Input and output for the interactive responses and reports are handledby the printer and keyboard control logic. Further, data from the camera18 and from the position sensors 26, 27 are handled by image acquisitioncontrol logic and position acquisition control logic, respectively.

The control logic of the software forms a operating shell which is usedby the analysis and measuring functions in blocks 82-96 to control thehardware of apparatus 11 to perform the particular function needed. Thesystem provides a patient labeling function 182 to identify theparticular tissue samples which are under study. Light calibration andposition calibration functions 84 and 86, respectively, are used todetermine a correct reference optical density for a particular field andthe location of that particular field with respect to a coordinateorigin. A mask acquisition function allows a nuclear mask to be formedso as to identify the total nuclear area of an image. A boundaryformation function 90 allows the operator to choose a reference levelagainst which the grey scale values of an image are compared for a maskimage or an antibody image. The cell data acquisition function 92provides a storage of the grey scale values of a specimen image. Autilities function 94 provides the needed auxiliary type programs forassisting in the primary functions of the image analysis. A reportgeneration function 96 is used for hardcopy production of analyzed andcompiled data from the system on the printer 38.

The support on which a specimen is viewed preferably is a transparentglass slide 14 as illustrated in FIGS. 4 and 5. Glass slides of arectangular shape come in standardized sizes such as 1" by 3" and suchcan be used with the following modifications. The slide 14 ispartitioned into two sections where in a first control section 56 arelocated control cell objects 40. In a second specimen section 58 thereare located specimen cell objects 52 which are to be measured for theircontent of estrogen receptors. The slide 14 further includes a border 54around the control section 56 for rapid identification purposes.Further, on some convenient location of the slide 14 is placed aidentifying mark 53. The mark 53, illustrated as a cross in FIG. 4, isof a predetermined optical density and can be used as a landmark foridentifying the coordinate origin for fields on the slide.

With respect to FIG. 5 there is illustrated a perspective view of thestructure which implements the filter selection apparatus 23. The filterselection apparatus 23 includes a mounting block 110 which has a viewingaperture centrally located therein for being disposed coincidentally inthe image acquisition path of the microscope 15 and analysis system 13of the apparatus 11. A slide 112 is positionable at three positions inthe mounting block 110 by means of a handle 114 guided in a slot 115.The slide 112 mounts three circularly shaped filter elements, eachcorresponding to one of the three positions of the handle 114. A firstelement 126 comprises a red filter which passes only light of a narrowbandwidth of wavelengths near 650±20 nanometers. A second elementcentered in the slide (not shown) is a neutral density filter and doesnot appreciably change the optical characteristics of light passingtherethrough. The third element 124 is a green filter which passes lightof a narrow bandwidth of wavelengths near 500±20 nanometers. Because ofthe narrow bandwidth of the filters 124, 126 the images passedtherethrough are essentially monochromatic.

When the aperture of block 110 is positioned in the light path of themicroscope 15, the operator has three choices for an image transmittedthrough apparatus 23 to the viewing optics 24 and the television camera18. Positioning the handle 114 at the far right position of the slot 115will cause a monochromatic filtering of the image with the red filter126, positioning the handle 114 in the center location of the slot 115will provide a substantially unfiltered image, and positioning thehandle 114 at the furthest left-hand side of the slot will produce amonochromatic filtering of the image with the green filter 124.

While the illustrated filter selection apparatus 23 is shown as manuallyselectable, it is evident that such apparatus could just as easily havebeen made automatically adjustable, such as by program control. Further,the function of the filter selection apparatus 23 could be integrated inmany of the other elements in the image analysis system 13 along theimage path. For example, a multiple color camera could be used toreplace camera 18. Additionally, prior to the image analysis, a digitalor analog filtering of the electrical image signal could beaccomplished. There are numerous methods by which the two monochromaticimages of the microscope field can be obtained, and that shown is meantto be exemplary not limiting.

The method of quantitating nuclear proteins includes providing specimencell objects in section 58 of slide 14 and staining them with an opticalenhancement factor which specifically binds to the nuclear protein. Thestain is then viewed with the image analysis system 13 to measure theoptical density of the stain for intensity measurement and to locate theareas in which stain is found for distribution measurement. Because theintensity of the staining relates to the quantity of the nuclearproteins, measurement of the different optical densities of the stainpermits a direct measurement of the quantity of the proteins. As anenhancement, control cell objects can be placed in section 56 of slide14 to provide a normalization or reference optical density for thestaining. Further, one or several counterstains can be used to furtherdistinguish among several features of the cell objects.

Preferably, in one particular embodiment, the staining method employs asensitive peroxidase-antiperoxidase technique for visualization ofestrogen or progesterone receptors in specimens through the use ofmonoclonal antibodies directed specifically against those receptors. Adiagrammatic representation of the process on the microscopic level isillustrated in FIGS. 7 and 8. Two portions of a human tumor specimencontaining a cell population from which the estrogen receptors are to bemeasured are placed on the two separate sections of the slide 14 andsuitably fixed thereto as by tissue adhesive. The separate sections arethen fixed in separate washes of formalin, methanol, and acetone, and,thereafter, treated with a blocking reagent to prevent nonspecificbinding of the subsequent reagents.

The part of the specimen cells to be measured is incubated with aprimary antibody, a monoclonal antibody (rat) to human estrogen receptorin the specimen portion 58 of slide 14. This antibody, as represented at128, binds specifically to the estrogen receptor sites ER of this tissueportion. The other portion of the specimen in the control section 56 ofthe slide 14 is incubated with a control, normal rat IgG, represented at130. The purpose of the control 130 is to evaluate the amount of bindingof the immunoperoxidase reagents in this technique to nonspecific sitesNS of the specimen to yield a background measurement.

Both sections 56 and 58 of the slide 14 are then incubated with abridging antibody, an anti-rat immunoglobulin (goat) illustrated at 132in both figures. The bridging antibody 132 binds to the rat antibody 128against human estrogen receptor in the specimen section 58 and to anybound normal rat IgG 130 in the control section 56.

A rat PAP complex 134 is added to both sections 56 and 58 of thespecimen and binds to the anti-rat Ig bridging antibody at 132. Afterthis step, a solution containing hydrogen peroxide and diaminobenzidine.4 HCl (DAB) is added to the specimen and control sections. The reactionof the peroxidase with hydrogen peroxide converts the bound DAB presentinto an insoluble reddish brown precipitate. The proportion of theprecipitate and its location are influenced by the binding positions ofthe PAP complex and, through the bridging and primary antibodies, thelocations and amounts of the estrogen receptors in the specimen.

The concentrations, timing, and chemical compositions of the reagentsused in this staining method are more fully described in the previouslyreferenced paper by McCarty Jr. et al. Preferably, the monoclonalantibody which is used to bind to the estrogen receptor sites is one ofthose developed at the University of Chicago and designated H222 sP2 andH226 sP2, and that which is used to bind to the progesterone receptor isone which is commercially available from Transbio Sarl 6 Rue Thiers,Paris France and designated mPRI.

It is further evident that instead of a single slide, as shown for thepreferred embodiment, separate slides can be used in the stainingtechnique for the control portion and the specimen portion. Whether asingle slide or two separate slides are used does not alter the qualityor amount of staining of the immunoperoxidase stain which binds to thereceptors of a specimen.

The DAB precipitate is then visualized by image analysis with apparatus11 to determine the quantitation of the estrogen receptors in thespecimen. In general, the brown precipitate does not transmit light welland will show up as dark areas in the cells of the specimen. The opticaldensity and hence pixel intensity will be related directly to the amountof DAB precipitated and to the quantity of estrogen receptor which hasbound the antibodies. To be able to more clearly visualize the nucleararea of each cell, a counterstain of methyl green is added. It isimportant to note that both the primary stain of DAB precipitate and thecounterstain of methyl green are specific to the nucleus of each cell.This means that debris and other cellular features will appear lighterin the microscope image and can be distinguished.

A dual filtering method is thereafter applied to distinguish the areasstained by the DAB and the areas stained by the methyl green. The redand green filters 126 and 124 respectively are used by moving handle 114to form monochromatic images of the cell objects which can be stored inthe apparatus 11. These images, one by the red filter and the other bythe green filter, are used to separate the primary stained areas fromthe nuclear areas, and to separate the nuclear areas from other cell orfield features.

The results and desirability of this dual filtering of a counterstainedcell image are more fully illustrated in FIG. 9. The percentage of lighttransmitted through the nuclei stained with methyl green is shown in thecurve A as a function of the wavelength of light. The percentage oftransmission of light for diaminobenzidine (DAB) is shown in curve B asa function of the wavelength of light. The bandwidth of wavelengths oflight passed by the green filter is illustrated in band C while thebandwidth of wavelengths of light passed by the red filter isillustrated in band D.

When an image of a counterstained cell population or specimen isfiltered with the green filter 124, substantially all of the areasstained with the methyl green will be invisible. This is because themethyl green curve A has a relative transmissive peak near thiswavelength band while the diaminobenzidine curve B is relativelynontransmissive in this band. Thus, the areas with primary DAB stain canbe separated from the nuclear areas. At the other extreme of the graph,the band D of the red filter is positioned at a place where just theopposite occurs. The methyl green curve A has a relativelynontransmissive valley in this bandwidth while the diaminobenzidinecurve B is also relatively nontransmissive. Thus, the nuclear areascontaining both the primary stain and the counterstain appear darkerthan other cell features and can be identified without a problem.

Because of the relative differences in light transmission between theprimary and counterstain in the two filtered bandwidths, the methylgreen stained area is enhanced during one filtering relative to otherareas of the cell, and the areas which have diaminobenzidine precipitateare enhanced relative to the methyl green areas during the otherfiltering. Thus, the nuclear areas of the cell objects are opticallyenhanced along with the areas having DAB precipitate.

While the implementation shows a convenient and advantageous method fordiscriminating between the two areas having counterstaining, it isrecognized that there are various other staining or optical enhancementmethods and filtering methods which can be used to optically enhance oneparticular area or feature over another cell feature. For thequantitation of the specific hormonal receptors shown (progesterone andestrogen receptors), what is important is to distinguish the nucleararea in total so that it can be compared to that nuclear area whichcontains receptors by the presence of the diaminobenzidine precipitate.

A functional flow chart of the method of analyzing and quantitatinghormonal receptors with the dual filtering technique is shown in FIG.10. In a first step, block 150, the specimen and control sections 56, 58of a slide 14 are stained as described previously with theimmunoperoxidase staining technique using the monoclonal antibodyagainst estrophilin, or the one against progesterone, for the specimensection, and a control antibody for the control section. The specimenand control sections 56, 58 then contain similar cell populations butwith different amounts of primary stain. The specimen and controlsections 56, 58 are thereafter counterstained with a methyl green stainin block 152.

The resulting preparation has green nuclei with varying degrees of browndiaminobenzidine precipitate. An unfiltered representation of a smallsection of a cell population image is shown in FIG. 11 where 200, 202,and 204 are green cell nuclei and where nuclei 202, and 204 have brownareas 206, and 208, respectively of DAB precipitate. Nucleus 200 doesnot contain any estrogen receptor and therefore does not have any DABprecipitate. Objects 210, 212, and 214 are various other cell featuresor debris from the tissue section.

A calibration procedure is done in blocks 154-162 in order to establishthree variables for the dual staining technique. The first variable isthe light level which is set in block 154. The calibration is done onthe control section 56 or, as mentioned previously, on a separatecontrol slide. Because grey scale images of optical density areanalyzed, the level of the background light is important as these valuesare representative of the % transmission of a reference level of light.Therefore, when calibrating the light level, the variable source 19 isadjusted at a clear portion of slide 14 until the instrument recognizesit is set at the correct reference level.

The control cell section is then imaged with the red filter in block 156so that background and other features of the cells can be eliminatedfrom the image and nuclear areas will stand out. The cell nuclearboundaries however must be distinguished to accurately display theareas, and this necessitates the formation of a reference grey level ornuclear boundary threshold.

A threshold is set for the recognition of positive grey levels for thenuclei in block 158. Positive nuclear grey levels are pixel levels abovewhich a nucleus boundary will be recognized from background. Due todifferent amounts of methyl green staining, to different reactions ofthe immunoperoxidase staining on different specimens, and to thedifferent quantity of nonspecific binding, the grey level representingthe nuclear boundaries of the specimen cells will vary. By identifying anuclear boundary reference level for the control cells, which come fromthe same specimen and are counterstained identically to the specimencells, a threshold for the measurement of any number of different fieldsin the specimen cells can be set.

After the nuclear boundary level has been stored, the apparatus 11 isused to make an image of the same field of control cells using the greenfilter in block 160. As was the case for the nuclear area, theboundaries of the DAB stained areas must be set by forming a thresholdor antibody boundary level.

A threshold is set for the recognition of positive grey levels for theantibody stains in block 162. Positive antibody grey levels are pixellevels above which the ER stain will be recognized from background.Thus, this threshold level is set to just above the highest grey levelof the control cell nuclei. This threshold is necessary to determine thecontribution of the nonspecific staining of the control cells and thecontribution of the counterstain, methyl green, to the total staindetected by the instrument. This antibody reference level permits thediscrimination between antibody negative stained cells and antibodypositive stained cells. After its determination, the antibody boundarylevel is stored in memory 73.

After the image analysis system 13 is calibrated and the tissue sampleis labeled, a nuclear mask image is formed of the specimen cells in thefield of interest using the red filter in block 164. The threshold setin the calibration operation is used for this function. The nucleibecause of the counterstaining and filtering stand out in the image asseen in FIG. 12 while the DAB areas are not visible. This filtered imageis stored as the nuclear mask image and is representative of the totalnuclear area of the particular field. The red filter is then changed tothe green filter and an antibody quantitation image is formed in block166 from the same specimen field. The filtered image, using the antibodyboundary level of the calibration step, has the brown DAB precipitateareas which stand out prominently from the rest of the cell features.

Thereafter, the nuclear mask image can be used to overlay the antibodyimage after it is filtered with the green filter. A representation ofthis combination is shown as FIG. 13 which illustrates that only thoseareas having precipitate 206, and 208 and which are within the nuclearareas are recorded.

A number of important parameters relating to the content of estrogenreceptor in the nuclei are then available by this technique. Ameasurement of the optical density of the areas 206 and 208 withinstrument 11 will give a direct indication of the quantity of estrogenreceptor. The intensity values from the image can be displayed in ahistogram in block 168. Further, a comparison of the nuclear area of theestrogen receptors, area 206 added to area 208, when compared as apercentage of total nuclear area, areas 200, 202 and 204 added together,will yield an indication of the distribution of estrogen receptor. Stillfurther, by combining the quantity measurement of estrogen receptor withthe distribution measurement, an indication of the prognosis ofendocrine therapy can be obtained.

Such parameters are available as an analysis display in block 170. Afterperforming measurement and analysis for one field, the operator of theapparatus 11 can stop the process, or can continue to measure morefields by moving the microscope 15 to the next field with adjustmentmeans 12, and 17 and starting over with block 164. The same calibrationvalues which were found in blocks 158, 162 are used for the boundariesof each field.

While one particular pairing of a chromagen and counterstain have beenpreferably indicated, it is evident that many others could be used tospecify a particular nuclear protein and amplify it for visualization,and pairing such with another counterstain for nuclear visualizationwould also be evident. The following table indicates other preferredchromagens and counterstains useful in the quantitation of nuclearproteins by image analysis. The list is not exhaustive and is suggestiveof other equivalents for nuclear protein quantitation involving dyeswith single immunoenzyme tagged monoclonal antibodies and counterstain,or paired immunoenzyme monoclonals utilizing different dyes.

    ______________________________________    EQUIVALENT CHROMOGENS AND NUCLEAR    COUNTERSTAINS FOR QUANTITATIVE IMAGE    ANALYSIS    Enzyme     Chromogen        Counterstain    ______________________________________    horseradish               DAB (brown)      methyl green    peroxidase 4-Cl-1-naphthol  hematoxylin               (grayish blue).sup.1                                nuclear fast red               alpha-naphthol   toluidine blue               followed by pyronin.sup.2               (reddish pink)               2, 2'-oxydiethanol-4-               chloronaphthol (black).sup.3    ______________________________________     .sup.1 Reaction products are soluble with organic solvents. Do not use     alcohol to dehydrate tissue sections. A water soluble medium, e.g. chrome     glycerin jelly, must be used to mount the cover slips. Method in Farr and     Nakane: J. Immunol. Meth. 47: 129-144, 1981.     .sup.2 Reaction product is soluble with organic solvents. Method in Taylo     CR, Burns J: J. Clin. Pathol. 27: 14-20, 1974.     .sup.3 Method in Van Rooijen N, Streefkerk JG: J. Immunol. Meth. 10:     370-383, 1976.

With the ability to assay not only the intensity of the estrogenreceptors but also their distribution in a cell population, a method isprovided by the invention for predicting favorable endocrine therapyresponse based upon a combination of these factors. A cell population ismeasured with the apparatus of the invention to determine the percentageof positive stained cells in the population and their average stainintensity. A combination score of these two factors is made according tothe formula: ##EQU2## where QIC=a quantitative immunocytochemical score;and

N=a scaling factor.

It has been determined empirically that when the scaling factor is 10, aQIC score of ≧18 corresponds with excellent sensitivity and specificityto other quantitative and semi-quantitative estrogen receptor assays.Particularly, a QIC score of ≧18 corresponds with a 98% sensitivityfactor and a 100% specificity factor when compared to a biochemicalassay of estrogen receptor setting an estrogen receptor rich thresholdat 10 Fmol./mg. of cytosol.

EXAMPLE

Two hundred cases of primary breast carcinoma accessioned from the DukeUniversity Endocrine Oncology Laboratory were used in a study to comparethe methods of the invention and apparatus 11 to two known estrogenreceptor tests, the biochemical assay and the subjective visual scoringby immunohistochemistry. These cases represented primary breast cancerspecimens with sufficient cancerous tissue for complete biochemical andimmunocytochemical analysis.

For each case, a biochemical estrogen receptor analysis was performed atthe time the tissue was first obtained. These consisted ofmulticoncentration titration analysis (dextran coated-charcoal analysis)and/or sucrose density gradient analysis of estrogen binding.

For immunocytochemical analysis cryostat sections of fresh frozen tissuewere fixed on slides in 3.7% formaldehyde-0.1 M phosphate bufferedsaline for 10 minutes followed by immersion in 100% methanol for 4minutes and acetone for 1 minute. Monoclonal antibody H222 sP2, preparedagainst human estrogen receptor protein was used.

The peroxidase anti-peroxidase method for immunocytochemicallocalization was performed as described by Sternberger. The blockingreagent was normal goat serum. The primary antibody (H222 sP2) which isa rat monoclonal antibody against human estrogen receptor, was used at aminimum concentration of 0.1≦g/ml. The bridging antibody was goatanti-rat immunoglobulin and the PAP complex was of rat origin. Controlslides consisted of an adjacent tumor section to that stained with theprimary monoclonal antibody in which the monoclonal antibody wasreplaced by normal rat immunoglobulin. The peroxidase localization wasdeveloped with diaminobenzidine-H₂ O₂. The slides were rinsed in runningtap water for 5 minutes and then dehydrated in serial alcohols toxylene, and coverslipped with Permount without counterstaining.

Computerized image analysis for one immunocytochemical assay was doneusing the apparatus illustrated in FIG. 1. Each case consisted of threeslides: a hematoxylin and eosin stained slide, a control slide and theprimary H222 sP2 stained slide. The control and primary slide werecounterstained with methyl green for this part of the study to aid inthe visualization of the cellular morphology. The coverslips wereremoved by soaking the slides in toluene. They were then placed insodium acetate buffer, rehydrated through an acetone series,counterstained with methyl green, rinsed with sodium acetate buffer,dehydrated with an acetone series to xylene and coverslipped withPermount.

The control slide was examined at 10× for an overall view of the tumorand counterstained intensity, and an area that was representative of thetumor with no tissue folding and minimal background staining chosen. Thecontrol antibody threshold was then determined at 40×. The primarysection for estrogen receptor was then evaluated. An overall screen fortumor and stain was accounted for at 10×. Five random fields at 40× werethen evaluated for chromagen-diaminobenzidine (DAB) intensity. If thestaining intensity was focally positive and negative, and heterogeneousstaining existed elsewhere in the section, a field representing thepositive and negative site as well as the heterogeneous area wasobtained and averaged.

In calculating the nuclear threshold the value taken was that in whichthe tumor cells were best represented by a pixelled image on the imagemonitor 37 with the control slide. If a pixelled image of the tumorcells on the primary slide was not complete, the threshold was broughtup or down accordingly. This was true for all 5 fields.

The antibody threshold was determined on the control slide by viewingthe image monitor 37 and increasing or decreasing the threshold until nopixelling was present and then brought up one step to account for thebackground staining on the primary slide. The primary slide was thenobserved and, if the histogram did not clearly separate the positive andnegative cells, the first field was adjusted accordingly. It was neveradjusted more than 3 units above or below the original control slideantibody threshold value. This was done only for the first field. Thefields were averaged to give the final calculations (percent positive,percent staining intensity and QIC score), and a histogram.

Biochemical assays were summarized as fmol of estrogen binding per mg ofcytosol protein.

The visual immunocytochemical localization was scored in asemi-quantitative fashion incorporating both the intensity and thedistribution of specific staining. The evaluations were recorded aspercentages of positively stained target cells in each of 4 intensitycategories which were denoted as 0 (no staining), 1+ (weak butdetectable above control), 2+ (distinct), and 3+ (intense). For eachtissue, a value designated the HSCORE was derived by summing thepercentages of cells stained at each intensity multiplied by theweighted intensity of staining:

    HSCORE=p.sup.i (i+1)

where i=1,2,3 and p^(i) varies from 0 to 100%. An HSCORE was assignedwith and without methyl green counterstain.

Computerized image analysis was complete on all of the cases and a QICscore was then generated using the formula above.

The comparison of the results of each type of test showed that thecomputer image analysis shows excellent correlation to the two otherrecognized tests. In particular, of the 200 initial cases, 100 were usedfor comparison against results of the other two tests as shown in thetable below.

    ______________________________________                  QIC ≧ 18                                 QIC < 18                  TP   FP        TN     FN    ______________________________________    VISUAL SCORING  74     1         24   1    BIOCHEMICAL     75     0         24   1    ANALYSIS    ______________________________________

where

TP=true positive

FP=false positive

TN=true negative

FN=false negative

Image analysis for sensitivity against visual scoring isTP/TP+FN=74/75=98%

Image analysis for specificity against visual scoring isTN/TN+FP=24/25=96%

Image analysis for sensitivity against biochemical analysis isTP/TP+FN=75/76=98%

Image analysis for specificity against biochemical analysis isTN/TN+FP=24/24=100%

The system program in general is a menu driven program which allows theoperator to interactively communicate with the image analysis system 13to produce the quantitation of nuclear protein by image analysis. Thesystem program displays a plurality of images or instruction screens onthe instruction monitor 62 which include menus from which to select thevarious functions needed for performing a quantitative nuclear proteinassay. FIG. 16 illustrates the screen architecture of the system and thepaths that the system takes between screens. Examples of two of thesystem screens, the nuclear mask screen A16 and the antibodyquantitation screen A20, which appear on the instruction monitor 62 arepictorially illustrated in FIGS. 14, and 15, respectively.

Returning to references in FIG. 16, the system program may be run bycalling it as an application program of the operating system A10.Selection of the system program by the operating system A10 produces theinitial screen A12 on the monitor 62. From the initial screen A12 theoperator can select a label/calibrate screen A14 or exit back to theoperating system. The operator may also exit back to the operatingsystem from the label/calibrate screen A14. While displaying thelabel/calibrate screen A14 the instrument can be calibrated to providethe background or reference light settings which will be used in themeasurement of the assay. Once the light calibration is complete theoperator can select the nuclear mask screen A16 which is used to form anuclear mask which will be later used in the assay technique.

One of the options in the nuclear mask screen A16 is to adjust thenuclear boundary which assists in forming the mask. Once the nuclearmask has been produced by the nuclear mask screen A16, the operator canselect the antibody quantitation screen A20 to actually do measurementsand generate reports. One of the options in the antibody quantitationscreen is to select the adjust antibody boundary screen A22 whichassists in the assay technique. Exits from the adjust antibody boundaryscreen A22 are to the antibody quantitation screen A20 which exits backto the nuclear mask screen A16.

In this manner an advantageous screen architecture is formed which canbe easily used and understood by the operator. This screen structurefacilitates the interactive measurement of the particular nuclearprotein under study. The instruction screens provide an interactive useof the digital imaging system which combines the power of the systemsoftware and hardware with the judgment and knowledge of the operator.The screen structure automates the assay task of nuclear proteinquantitation while still permitting the operator to selectively choosethe input data and control the process to a considerable degree.

Each screen A12-A22 contains a menu of the functions permitted for usewhile that particular screen is being displayed on the instructionmonitor 62. The function that the system is to currently execute ischosen by the operator with a cursor movement method using the standardcursor control keys of keyboard 36. While a particular screen is beingshown on the monitor, the cursor movement keys are operable to positionthe cursor next to a particular function of the menu on that screen.While the cursor highlights the function by its position, the operatormay select the function for execution by pressing the enter key.

The initial screen A12 contains the main menu for the nuclear proteinquantitation program. The main menu illustrated in FIG. 17 has twochoices which are to select either (1) a label/calibrate function, or(2) an exit function. Selection of the exit function will return controlto the operating system A10 so that the processor may execute otherapplication programs or be used as a general purpose computer. Theselection of the label/calibrate function will cause the program tochange the display on instruction monitor 62 from the initial screen A12to the label/calibration screen A14. The label/calibrate screen containsa number of functions which will allow the operator to calibrate thelevel of background light from source 19, type in patient information,set the xy coordinates for the present field, and to access the nuclearmask screen A16. An exit function will cause the exit of the systemprogram back to the operating system.

The selections of the label/calibrate menu are shown in FIG. 18 andinclude the choices of (1) label, (2) set-light, (3) set-xy, (4) nuclearmask, or (5) exit. The label function A32 allows the operator to enterinformation regarding patient identification and accession number by aninteractive editing routine. Because this information is general to theestrogen receptor assay, it will appear on every screen A14-A22. Theoperator presses the enter or the escape key to exit the labeloperation. Pressing the enter key will save to memory 73 any changeswhich were made to the information during the function, and pressing theescape key will cause the system to ignore any changes which were made.The information which is input during the label function A32 will not besaved when the system program is exited to the operating system.

The set-light function A34 calculates the average light level (greyscale value) for the current image and allows the operator tointeractively adjust the level by variation of the intensity of source19 until it is at a correct reference level. The operator views thefield of the slide 14 on the image month 37 and positions the slide withadjustment means 12 and 17 (FIG. 1) until a clear field is found. Theset-light function A34 must be successfully performed at least once tobe able to select the nuclear mask function. The set-light function A34is successful when the current image is blank and the light level is setbetween 129 and 131, preferably 130 for the most accurate results. Theimage acquisition control logic will perform noise subtraction, if theset-light function A34 has been successfully executed.

The set-xy function A36 is utilized by the operator to set the currentimage location as the origin of an xy coordinate system for the slide.Thus, the set-xy function A36 should be used every time a new slide 14is selected. If the set-xy function is not executed then the xy functionA56 (FIG. 19) in the program will not be able to be selected by theoperator. Further, if the microscope platform 51 is being moved when theset-xy function A36 is in progress, then the function may not besuccessfully performed. A message will appear on the screen A14 to letthe operator know if the set-xy function has been successfully executed.Upon a non-successful execution of the set-xy function, the operator hasto reselect the function.

Selection of the nuclear mask function A38 will change the display onthe instruction monitor 62 from the label/calibrate screen A14 to thenuclear mask screen A16 which is shown in FIG. 14. The nuclear maskscreen A16 contains a menu having selections which allows the operatorto adjust the nuclear boundary, display the nuclear area, specify anarea of an image to analyze, display the current light level, displaythe xy coordinates of the present field, access the antibodyquantitation screen, or clear the data from the storage. The onlyrequirement before using the nuclear mask function is that the set lightfunction A34 of FIG. 18 must have been successfully executed previously.

The exit function of this part of the program allows the operator toexit the system program by either selecting the escape key or the exitfunction. When the exit operation is specified, the user will be askedto confirm his decision to exit. To accept the confirmation the operatorselects the yes key and to reject the confirmation, the operator selectsthe no key or presses the escape key.

A description of the nuclear mask functions will now be more fullydetailed with reference to FIG. 19. The adjust nuclear boundary functionA44 will change the display on the monitor from the nuclear mask screenA16 to the adjust nuclear boundary screen A18. The adjust nuclearboundary screen A18 contains a menu for selection of the functions thatallows an operator to change the nuclear boundary. The adjust nuclearboundary function must be used every time that the operator wants tomeasure data from an image field. Thus, every time a new image isselected for measurement, the adjust nuclear boundary function A44 mustbe selected and set before the display nuclear area function A46 or theantibody quantitation function A48 can be selected.

Selection of the display nuclear area function will cause the imagemonitor 37 to display the nuclear area on the screen. The programspecifies a specific window in the image wherein the nuclear area inthat specific window will be displayed in yellow and everything else inthe image will be displayed in white. The yellow and white image will becleared from monitor 37 whenever the next function is selected. Thisfunction gives a visual image for comparison with the calculation ofnuclear area.

Selection of the antibody quantitation function A48 will cause theinstruction monitor 62 to change the display from the nuclear maskscreen A16 to the antibody quantitation screen A20. The antibodyquantitation screen A20 illustrated in FIG. 16 contains a menu havingfunctions that allow the user to measure an image, display a histogramof that data, smooth the histogram data, adjust the antibody boundary,change the histogram scale, clear the data, select the next image field,and to return to the label and calibration screen.

The selection of the window function A50 allows the operator to specifywhich areas of the current image to store for the mask image. The areawhich is to be stored can be shrunk, expanded or moved to a particulararea in the field of interest. Initially, the window size is set to thewhole image accept that part of the image which lies outside a window 64(FIG. 1) displayed on the image monitor 37. The window function A50allows the operator to adjust the size of window 68 and to move thewindow. When first entered, the window function A50 will be in agrow/shrink mode. While in this mode the operator is allowed to increaseor decrease the window size. The following table lists the keys onkeyboard 36 which the operator presses in order to change the windowsize:

    ______________________________________    KEY       ACTION    ______________________________________    0         alternate between large and small increments    5         alternate between the shrink and grow modes    1         increase or decrease the left and bottom              sides of the window    2         increase or decrease the bottom side of the              window    3         increase or decrease the right and bottom              sides of the window    4         increase or decrease the left side of the              window    6         increase or decrease the right side of the              window    7         increase or decrease the left and top sides              of the window    8         increase or decrease the top side of the              window    9         increase or decrease the right and top sides              of the window    ESC       alternate between the grow/shrink mode and              the move mode    ______________________________________

If the escape key is pressed by the operator while in the grow/shrinkmode of the window function A50, the mode will change to a move mode.While in the move mode, the operator will be able to move the windowanywhere within the field of view. The following table lists the keysthat the operator can select to move the window:

    ______________________________________    KEY       ACTION    ______________________________________    0         alternate between large and small increments    1         move the window in the down-left direction    2         move the window in the down direction    3         move the window in the down-right direction    4         move the window in the left direction    6         move the window in the right direction    7         move the window in the up-left direction    8         move the window in the up direction    9         move the window in the up-right direction    ESC       alternate between the grow/shrink mode and              the move mode    ______________________________________

If the operator wishes to adjust the window 68 for the current field ofimage, the window function must be selected before the adjust nuclearboundary function. The window size will be reset to normal every timethe clear function A58 is selected in the nuclear mask screen, or whenthe antibody quantitation screen is exited.

The selection of the focus function A52 in the nuclear mask menu willprovide color enhancement for the image on image monitor 37 so that theoperator can perform more precise focusing of the image. The colorenhanced image will disappear when any one of the following functions isselected: adjust nuclear boundary A44, display nuclear area A46,antibody quantitation A48, check light A54, focus A52, and window A50.

The check light function A54 will calculate the present average lightlevel of the current field on the image monitor 37. If the light levelis not the calibrated reference level between 129-131, the operator canreturn to the label calibrate screen to reset the level.

Selection of the xy function A56 for the nuclear mask screen A16 causesthe x, y coordinates of the current image to be displayed. The xyfunction A56 displays the x, y coordinates of the current image relativeto the origin which was set in the set-xy function A36 (FIG. 18). Thecoordinates will be continually displayed until the user presses anotherkey. The xy function A56 is used to help locate fields of view whichhave previously been observed and marked for further analysis.

The selection of the clear function A58 will cause an erasure of all theimage data stored for the session. The window size and placement willalso be reset. After the clear function A50 has been selected by theoperator, he will be requested to confirm the operation. To accept theconfirmation, the operator selects the yes key and to reject theconfirmation, the operator selects the no key or presses the escape key.

The exit function A60 in the nuclear mask screen A16 A16 will cause theinstruction monitor 62 to change the display from the nuclear maskscreen to the label/calibrate screen A14. Pressing the escape key is thesame as selecting the exit function.

The functions of the antibody quantitation screen A20 will now bereferenced to the antibody quantitation menu of FIG. 21. The selectionof the measure function A80 allows the operator to measure the nuclearstain values that are inside of the window of the current field orimage. After the first measure operation is completed, a histogram (FIG.16) of the nuclear data of the measured image will be displayed on theantibody quantitation screen A20 of the instruction monitor 62 and theword "single" will appear on the bottom of the screen. The word singleindicates to the operator that the displayed histogram belongs to theimage presently being measured. In addition, the positive percent, thenuclear area, and the positive stain intensity of the measured image isdisplayed. The measure function A80 can be selected by the operator anynumber of times for the current image as long as the merge function A84has not been selected.

If the merge function A84 is selected, the measure function A80 will notlonger apply just to the current image. The antibody quantitation screenA20 must be exited, and reentered in order to use the measure functionA80 in the single mode again. If the measure function A80 is selectedbut the merge function A84 is not used to save the data, then the datathat was calculated in the measure operation will be lost when theantibody quantitation screen A20 is exited.

Selection of the smooth function A82 allows the operator to smooth roughareas of the displayed histogram on the instruction monitor 62. Thehistogram can be smoothed to display from 1-9 peaks. After the smoothfunction A82 is selected, the cursor will move to the location on thescreen A20 where the user can type in a smoothing value from keyboard36. The smoothing value must be in the range 0-9. A smoothing value ofzero will display the raw histogram on the screen while a smoothingvalue of 9 is the maximum amount of smoothing available. To execute thefunction and smooth the histogram, the operator presses the enter keyafter typing in the smoothing value. The operator can abort thesmoothing operation before it is executed by pressing the escape key.

The selection of the merge function A84 allows the user to accumulatedata from different specimen fields. The merge function A84 can only beselected after the measure function A80 has been used at least once forthe current image. The merge function A84 will display in the histogramof all of the data that has been accumulated by the previous selectionsof the merge function A84. The scale of the histogram will be that ofthe measure function. The positive percent, nuclear area, and positivestain intensity of the accumulated data will also be displayed on theinstruction monitor 62. The word total will be displayed at the bottomof the screen to indicate to the user that the displayed histogramrepresents all the data that was collected using the merge function A84.

The display nuclear area function A86 in the antibody quantitationscreen A20 functions similar to that function described for the nuclearmask screen A16.

Selection of the adjust antibody boundary function A88 will change thedisplay on the instruction monitor 62 from the antibody quantitationscreen to the adjust antibody boundary screen A22. The adjust antibodyboundary screen A22 provides a function that will allow the user toadjust the antibody boundary to a desired level.

Selection of the next field function A90 in the antibody quantitationscreen A20 permits the operator to select another image to measure. Thedisplay on the instruction monitor 62 will change from the antibodyquantitation screen A20 to the nuclear mask screen A16. Selection ofthis operation will also reset the window size.

When the operator selects the comment function A92, the apparatus 11allows him to type in a comment on the antibody quantitation screen A20in a particular area. The comment will appear only on the antibodyquantitation screen. After the function is selected, the cursor willmove to the location where the comment can be typed from keyboard 36. Anediting routine is entered and the comment received. To exit theoperation, the operator presses the enter or escape key. If the operatorpresses the enter key the system will save any changes that were made inthe comment area. Otherwise, pressing the escape key will command thesystem to ignore any changes that were made to the comment area.

The scale function A94 allows the operator to change the scale on thehorizontal axis of the histogram being displayed in the antibodyquantitation screen A20. There are three scales to choose from, 0-47,0-94, and 0-141 which represent the grey scale values of the pixels ofthe image. If the scale function A94 is selected when the current scaleis 0-47, then the new scale will be 0-94. Similarly, if the scalefunction A94 is selected when the current scale is 0-94, then the newscale will be 0-141, etc. If the current scale is 0-47, then the lasthistogram column D will contain the histogram data for the grey scalevalues 48-255 and the first three columns A-C equally divides the restof the scale. If the current scale is 0-94, then the last histogramcolumn will contain the histogram data for the grey scale values 95-255.If the current grey scale is 0-141, then the last histogram column willcontain histogram data for the grey scale values 142-255.

The selection of the xy function A96 will cause the system to display onthe instruction monitor 62 the x, y coordinates of the current field orimage. The xy function of this screen operates similarly to thatdescribed for the nuclear mask screen A16.

If the operator selects the label/calibrate function A98, the systemwill change the display from the antibody quantitation screen A20 to thelabel/calibrate screen A14. Selection of this function A98 will alsoreset the window size.

If the clear function A100 is selected by the operator, the system 13will clear all the data presently acquired for the image and the windowsize will also be reset. After the clear function A100 has beenselected, the operator will be requested to confirm the clear operationby a prompt on the instruction monitor 62. To accept the confirmationthe operator will select the yes key and conversely, to reject theconfirmation the operator will select the no key or press the escapekey. If the yes key is selected by the operator, the display on theinstruction monitor 62 will change from the antibody quantitation screenA20 to the nuclear mask screen A16.

Selection of the exit function A102 causes the system 13 to change thedisplay on the instruction monitor 62 from the antibody quantitationscreen A20 to the nuclear mask screen A16. Pressing the escape key isthe same as selecting the exit function A102. The window size will alsobe reset upon the execution of the exit function A102.

The functions available for the adjust antibody boundary screen A22 willnow be more fully described with reference to the menu of FIG. 22.Selection of the switch function A72 will cause the image displayed onthe image monitor 37 to alternate between a color enhanced image and theoriginal image. However, image color enhancement, the focus function A52(FIG. 19) must previously have been selected.

The set step size function A74 allows the operator to change the amountof grey scale units that the antibody boundary will change when one ofthe cursor keys is selected. The value chosen must be in the range of0-128. After the set step size function A74 is selected, the cursor willmove to the location on the adjust antibody boundary screen where theuser can type in the new step size value. To exit from the set step sizefunction A74, the operator presses either the enter or the escape keys.Pressing the enter key will save the step size change, while pressingthe escape key will ignore any change that was made. As a default valuethe program initially sets the step size value equal to one.

Thereafter, repetitions of an up arrow key will increase the antibodyboundary by the value of the step size for each press and the fieldunder consideration with changed antibody boundary value will bedisplayed on the image monitor 37. Selection of the down arrow key willdecrease the antibody boundary value by the value of the step size foreach press. The new antibody boundary value will be displayed on theinstruction monitor 62. Further, the image of the field on the monitor37 will reflect the changes made in the boundary value.

Selection of the exit function A76 changes the display from the adjustantibody boundary screen A22 to the antibody quantitation screen A20.Pressing the escape key is the same as selecting the exit function.

FIG. 15 illustrates the report generation stage of the analysis whichtakes the form of a histogram. The histogram has as its ordinate axisthe percentage of total nuclear area and as its abscissa axis theintensity of the stain. Thus, each bar of the histogram illustrates thepercentage of the total nuclear area that was measured for a particularstain intensity. The stain intensity axis is divided up into fourseparate areas A-D with each having a greater of the stain intensityvalues, for example, area A corresponds to intensity values 0-64, area Bcorresponds to intensity values 65-128, area C to values between129-192, and area D to values between 193-256. The sum of the percentageof total nuclear area for the stain intensities of each block A-D isdisplayed centered in the block. Further, a vertical marker is used tovisually differentiate positive stain values from negative stain values.

A summary of the measurements made are displayed in the measurementreport area next to their titles. The "measure count" indicates how manyspecimen fields have been measured to obtain the present data. The"positive percent" indicates the total percentage of nuclear area withpositive stain, i.e., that area pictured right of the vertical marker inthe histogram. The "nuclear area" is the total nuclear area of all thefields measured for the present data and is given in μm². The "positivestain intensity" is the average grey scale value arrived at taking intoaccount all values of intensity for the positive stain values.

While a preferred embodiment of the invention has been illustrated, itwill be obvious to those skilled of the art that various modificationsand changes may be made thereto without departing from the spirit andscope of the invention as defined in the appended claims.

What is claimed is:
 1. A method for measuring a nuclear antigen in atissue sample containing cells with an immunohistochemical techniqueusing an antibody to said nuclear antigen and a microscopic digitalimage measuring means, said method comprising the steps of:staining thesample with an immunohistochemical technique to optically enhance and tostain nuclear antigens with a first spectral stain material which varieswith the quantity of the nuclear antigen contained in the sample,optically enhancing the sample with a second spectral stain materialwhich optically enhances nuclear material in the cells, measuring theoptically-enhanced nuclear material with a microscopic digital imagemeasuring means at a first wavelength range which allows identificationof all of the enhanced nuclear material, measuring theoptically-enhanced antigen with the microscopic digital image measuringmeans at a second wavelength range which substantially excludes theoptically-enhanced nuclear material, and quantitating the nuclearantigen using the measurements made.
 2. A method for measuring thequantity of a nuclear antigen as set forth in claim 1 wherein the stepof staining further includes:staining said sample withperoxidase-antiperoxidase complex and a monoclonal antibody to estrogenreceptors thereby producing different quantities of a precipitate ofdiaminobenzidine having varying optical densities in dependence on theamount of estrogen receptor in the nuclear material in said sample.
 3. Amethod for measuring the quantity of a nuclear antigen as set forth inclaim 2 wherein said step of staining the sample further includes asecond stain step ofcounterstaining said sample with a second imageenhancement factor which enhances all nuclear material of said sample.4. A method for measuring the quantity of a nuclear antigen as set forthin claim 3 wherein said step of counterstaining includes:staining saidsample with methyl green.
 5. A method for measuring the quantity of anuclear antigen as set forth in claim 3 wherein said step of measuringthe nuclear material further includes:measuring all nuclear material insaid sample by filtering an image of said sample with a first filter toproduce an enhanced first filtered image which only represents the areaof the total nuclear material of said sample.
 6. A method for measuringthe quantity of a nuclear antigen as set forth in claim 5 wherein saidstep of measuring for the nuclear antigen further includes:filtering theimage of said sample with a second filter to produce an enhanced secondfiltered image of nuclear antigen areas of said total nuclear area whichare stained.
 7. A method for measuring the quantity of a nuclear antigenas set forth in claim 6 wherein said step of filtering the image of saidsample includes:filtering the image of said sample with said firstfilter having a bandpass to light substantially in the 620 nanometerrange to provide a first filtered image.
 8. A method for measuring thequantity of a nuclear antigen as set forth in claim 7 wherein said stepof filtering the image of said sample includes:filtering the image ofsaid sample with said second filter having a bandpass to lightsubstantially in the 500 nanometer range to provide a second filteredimage.
 9. A method for measuring the quantity of a nuclear antigen asset forth in claim 8 wherein said step of filtering includes:combiningsaid enhanced first filtered image with said enhanced second filteredimage to exclude image areas not included in both said enhanced firstfiltered image and said enhanced second filtered image.
 10. A method formeasuring the quantity of a nuclear antigen as set forth in claim 8wherein said step of filtering includes:combining said enhanced firstfiltered image with said enhanced second filtered image to exclude imageareas of said enhanced second filtered image which are not included insaid enhanced first filtered image.
 11. A method for measuring thequantity of a nuclear antigen as set forth in claim 5 wherein said stepof filtering the image of said sample with a first filter furtherincludes:comparing the first filtered image with a first thresholdoptical density and setting areas in said image with an optical densitybelow said first threshold to a background optical density.
 12. A methodfor measuring the quantity of a nuclear antigen as set forth in claim 11wherein said step of measuring optical density includes:determining afirst area of said enhanced first filtered image which has an opticaldensity in excess of said first threshold.
 13. A method for measuringthe quantity of a nuclear antigen as set forth in claim 6 wherein saidstep of filtering the image of said sample with a second filter furtherincludes:comparing said second filtered image with a threshold opticaldensity and setting areas in said image with an optical density belowsaid threshold optical density to a background optical density.
 14. Amethod for measuring the quantity of a nuclear antigen as set forth inclaim 13 wherein said step of measuring optical densityincludes:determining an area of said second filtered image which has anoptical density in excess of said threshold optical density.
 15. Amethod for measuring the quantity of a nuclear antigen as set forth inclaim 14 wherein said step of measuring optical density furtherincludes:determining the proportion that said second area is of saidfirst area.
 16. A method for measuring the quantity of a nuclear antigenas set forth in claim 15 wherein said step of determining a second areaincludes:calculating said threshold optical density from a controlsample which is similarly stained as said first-mentioned sample.
 17. Amethod in accordance with claim 1 including the further steps of:formingseparate optically-enhanced images of the nuclear antigen and thenuclear material and then electronically combining these separate imagesand presenting them to a user for viewing as a combined image showingboth the image-enhanced nuclear antigen and the nuclear material.
 18. Amethod in accordance with claim 1 in which step of quantitatingcomprises counting the cells and counting as positive those cells whichexhibit the antigen and reporting the percentage of positive cells ofthe total cells counted.
 19. A method in accordance with claim 1including the steps of measuring the first optically-enhanced nuclearantigen at a bandwidth wavelength range at which the first spectralstain material has a substantial transmittance and at which the secondspectral stain material is substantially nontransmissive.
 20. A methodin accordance with claim 19 in which the step of measuring the opticaldensity of the optically-enhanced image includes measuring at abandwidth at which the spectral stain has approximately 100%transmittance.
 21. A method in accordance with claim 19 wherein thenuclear antigen and nuclear material are stained with two differentspectral materials and measuring one of the spectral materials where theother materials has the greatest transmission.
 22. A method of providinga quantitative immunocytochemical measurement of a nuclear antigen in acarcinoma, said method comprising the steps of:providing a tissuespecimen having a cell population of the carcinoma; staining said cellpopulation with an immunological first stain which specificallyidentifies an antigen in the cell's nucleus; measuring the percentage ofthe cell population stained with said first stain and with a microscopicdigital image measuring means at a first wavelength; staining said cellpopulation with a counterstain which enhances all nuclear material ofsaid sample; measuring the intensity of said counterstain applied to thecell population with the microscopic digital image measuring means at asecond wavelength; and forming a quantitative immunocytochemicalmeasurement utilizing both the percentage of the cell population stainedand the intensity of the stain for the cell population.
 23. A method asset forth in claim 22 wherein said step of staining includes:stainingsaid cell population with a peroxidase-antiperoxidase complex andincluding a monoclonal antibody against estrophilin; and counterstainingsaid cell population with methyl green.
 24. A method as set forth inclaim 22 wherein said step of measuring stain intensityincludes:measuring the optical density of the area stained with aprimary stain which is in excess of a threshold value.
 25. A method asset forth in claim 22 wherein said step of measuring the percentage of acell population stained includes:measuring the total nuclear area ofsaid cell population; measuring the nuclear area of said cell populationstained with a primary stain; and calculating the percentage that saidprimary stained nuclear area is of said total nuclear area.
 26. A methodof analysis of estrogen or progesterone receptors in tumors in a humanbreast carcinoma, said method including the steps of:staining cells in acarcinoma cell population by an immunocytochemical technique tooptically enhance and to specifically identify the nuclear antigensestrophilin or progesterone with a first spectral stain material whichvaries with the quantity of said nuclear antigen, optically enhancingthe sample with a second spectral stain material which opticallyenhances nuclear material in the cells, measuring the optically-enhancednuclear material with a digital image measuring means at a firstwavelength range which allows identification of all of the enhancednuclear material, measuring the optically-enhanced antigen with themicroscopic digital image measuring means at a second wavelength rangewhich substantially excludes the optically-enhanced nuclear material,and quantitating the estrophilin or progesterone antigen using themeasurements made, providing a predetermined value above which tumorsare classified as an estrogen or progesterone receptor rich tumor andbelow which tumors are classified as an estrogen or progesteronereceptor poor tumor; and reporting a quantitative analysis based onmeasured value relative to the predetermined value for the tumors.
 27. Amethod for measuring the quantity of a specific nuclear antigen in acell population taken from a human breast carcinoma with a microscopicdigital image measuring means, said method comprising:obtaining a cellpopulation sample from a human breast carcinoma; staining said samplewith an immunohistochemical technique using an antibody against thespecific nuclear antigen, said immunohistochemical technique includingan image enhancement factor such that the optical density of said samplevaries with the quantity of nuclear antigen contained within saidsample; staining said sample with at least one other image enhancementfactor which is specific for the nucleus of the cells in said sample;measuring the optical density of the nuclear antigen in said sample withthe microscopic digital image measuring means; differentiating thenuclear area of the specific nuclear antigen stained with said opticalenhancement factor and measuring these differentiated stained areas withthe microscopic digital image measuring means; and determining whatpercentage the stained nuclear antigen is of the total nuclear area. 28.A method for measuring the quantity of a specific nuclear antigen as setforth in claim 22 wherein:the specific protein is estrogen receptor. 29.A method for measuring the quantity of a specific nuclear antigen as setforth in claim 29 wherein:said monoclonal antibody is H222 sP2 or H226sP2.
 30. A method for measuring the quantity of a specific nuclearantigen as set forth in claim 28 wherein:the specific nuclear antigen isprogesterone receptor.
 31. A method for measuring the quantity of aspecific nuclear antigen as set forth in claim 31 wherein:saidmonoclonal antibody is mPRI.
 32. A method for measuring the distributionof a specific nuclear antigen in a tissue sample with a microscopicdigital image measuring means, said method comprising:obtaining a tissuesample from a carcinoma; staining said sample with animmunohistochemical technique using an antibody against the specificnuclear antigen, said immunohistochemical technique including an imageenhancement factor such that the optical density of said sample varieswith the quantity of nuclear antigen contained within said sample;staining said sample with at least one other image enhancement factorwhich is specific for the nucleus of the cells in said sample; measuringthe optical density of the nuclear antigen in said sample with themicroscopic digital image measuring means; separating the nuclear areaof the specific nuclear antigen stained with said optical enhancementfactor and measuring these separated stained areas with the microscopicdigital image measuring means; and determining what percentage thestained nuclear antigen is of the total nuclear area.